1. Field of the Invention
The subject of the invention is a culture medium for Haemophilus influenzae type b in which the source of protein nitrogen comprises at least one plant peptone and in which the heme source consists of protoporphyrin IX. The invention also relates to a method for producing polyribosyl ribitol phosphate (PRP) used in the manufacture of a vaccine against Haemophilus influenzae type b meningitis.
2. Summary of the Related Art
The capsule is the major factor of virulence of Haemophilus influenzae type b strains. It is a polysaccharide consisting of a succession of repeating units of ribosyl ribitol phosphate. The expression polyribosyl ribitol phosphate (PRP) or capsular polysaccharide type b is used interchangeably to denote the Haemophilus influenzae type b capsule.
Haemophilus influenzae type b populations are often heterogeneous; capsulated bacteria coexist with noncapsulated bacteria. The noncapsulated bacteria have lost their capacity to express the capsule following genetic mutations which occur spontaneously. According to Hoiseth et al. (Infectious and Immunity (1985), 49: 389-395), the loss of the expression of the capsule occurs at a frequency of 0.1 to 0.3% at each bacterial generation. At the genetic level, the cap locus has been shown to be involved in the expression of the capsule at the surface of these bacteria (Kroll, J. S., et al., J. Bacteriol. (1988) 170: 859-864). The capsulated bacteria have a cap locus which contains at least two copies of an 18 Kb gene. The noncapsulated bacteria no longer have an 18 Kb gene or only a single copy of this gene. To identify the capsulated bacteria, the test of agglutination on a slide of bacteria in the presence of an anti-PRP antibody is usually used or molecular biology techniques which characterize the cap locus are used.
Vaccines based on PRP, or PRP covalently linked to a carrier protein, are used to prevent Haemophilus influenzae type b infections. To manufacture these vaccines, it is necessary to produce large quantities of bacteria in large volumes of culture medium from which PRP is extracted and then purified. Nevertheless, the ease with which the capsulated Haemophilus influenzae type b bacteria revert to noncapsulated forms can constitute a stumbling block for the production of PRP.
For the industrial production of PRP, culture media are generally used which are based on animal peptones which represent the principal source of protein nitrogen supplemented with yeast extract, glucose, hemin, β-NAD and inorganic salts. By way of example, the production medium described in U.S. Pat. No. 4,459,286 is mentioned.
Because of the risks linked to BSE, it is sought to replace products of animal origin and more particularly products of human or bovine origin with products offering better biological safety.
Carty et al. (in Dev. Indust. Microbiol. 26: 763-767 (1985)) have shown that animal peptones could be replaced by soybean peptone for the production of PRP. The composition per liter of this medium (MP medium) is the following: soybean peptone: 10 g; yeast extract: 10 ml; NaCl: 5 g; K2HPO4: 2.5 g; Na2HPO4: 3.3 g; dextrose: 5 g; hemin chloride: 10 mg; NAD: 10 mg.
Takagi et al. (J. Chem. Tech. and Biotech 81: 182-188 (2006) have sought to optimize the composition of the Carty medium (MP medium). They have shown that the PRP concentration in the culture medium could be increased by 70% to reach 0.25 g/l when the hemin and β-NAD concentrations were increased. To increase the production of PRP, it therefore appears to be necessary to increase the cofactor (hemin and β-NAD) concentrations, which are necessary for the growth of Haemophilus influenzae type b.